During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F. ananassa and F. vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F. ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphis fabae and A. ruborum, further mechanically transmitted to Nicotiana occidentalis 37B and sub-inoculated to N. benthamiana, N. benthamiana DCL2/4i, N. occidentalis 37B, and Physalis floridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F. vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphon malvae, A. fabae, and A. ruborum were shown to transmit StrV-1 under experimental conditions.

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Čačanská raná.

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3' end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

We collected samples from black, red and white currants showing symptoms of blackcurrant reversion disease (BRD) and full blossom disease (FBD), cultivated in the Czech Republic. Blackcurrant reversion virus (BRV) was detected in all symptomatic plants. After amplification, a substantial part of the 3′ non-translated region (3′-NTR) of RNA2 of 15 new isolates of BRV was sequenced and compared with sequences available in the literature and GenBank. We did not find significant sequence diversity among isolates associated with either FBD or BRD. BRV was graft-transmitted from FBD infected red currant to black currant where symptoms of BRD were observed. Further sequence analysis of BRV isolates resulted in a phylogenetic tree with four branches, each consisting of six to nine isolates. No correlation with geographic origin was visible on the tree as isolates from various countries occurred in all four branches. We also found no correlation between the host and the topology of the tree: most of black currant isolates occurred in branches 3 and 4, but also occurred in branches 1 and 2. Only one white currant and one red currant isolate occurred in branches 3 and 4, respectively. The sequence identity of the Czech isolates in this region ranged from 91.9 to 99.8%. The 17 plant species growing within and in the close vicinity of the BRDinfested plantation were tested negative for BRV by RT-PCR as natural hosts of BRV. BRV was successfully transmitted by mechanical inoculation from black currant to Nicotiana occidentalis and N. tabacum cv. Xanthi, the latter being a new host for BRV. The infection was confirmed by PCR and sequencing.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

During high throughput sequencing (HTS) of leaves from a symptomatic red clover plant, a new RNA virus, tentatively named red clover nepovirus A (RCNVA), was discovered. The complete genomic sequence was determined and characterized. Particularly noteworthy was that RCNVA shares high sequence identities in RNA1 with a group of phylogenetically related nepoviruses while homologies in the RNA2 segments are markedly lower. Based on the genomic organization and phylogenetic attributes, RCNVA should be classified as a novel virus of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales).